Eukaryotic cell division proceeds through a highly regulated cell cycle comprising consecutive phases termed G1, S, G2 and M. Disruption of the cell cycle or cell cycle control can result in cellular abnormalities or disease states such as cancer which arise from multiple genetic changes that transform growth-limited cells into highly invasive cells that are unresponsive to normal control of growth. Transition of normal cells into cancer cells can arise though loss of correct function in DNA replication and DNA repair mechanisms. All dividing cells are subject to a number of control mechanisms, known as cell-cycle checkpoints, which maintain genomic integrity by arresting or inducing destruction of aberrant cells. Investigation of cell cycle progression and control is consequently of significant interest in designing anticancer drugs. (Flatt, P. M. and Pietenpol, J. A. Drug Metab. Rev., (2000), 32(3–4), 283–305; Buolamwini, J. K. Current Pharmaceutical Design, (2000), 6, 379–392).
Accurate determination of cell cycle status is a key requirement for investigating cellular processes that affect the cell cycle or are dependent on cell cycle position. Such measurements are particularly vital in drug screening applications where:
i) substances which directly or indirectly modify cell cycle progression are desired, for example, for investigation as potential anti-cancer treatments;
ii) drug candidates are to be checked for unwanted effects on cell cycle progression; and/or
iii) it is suspected that an agent is active or inactive towards cells in a particular phase of the cell cycle.
Traditionally, cell cycle status for cell populations has been determined by flow cytometry using fluorescent dyes which stain the DNA content of cell nuclei (Barlogie, B. et al, Cancer Res., (1983), 43(9), 3982–97). Flow cytometry yields quantitative information on the DNA content of cells and hence allows determination of the relative numbers of cells in the G1, S and G2+M phases of the cell cycle. However, this analysis is a destructive non-dynamic process and requires serial sampling of a population to determine cell cycle status with time. Furthermore, standard flow cytometry techniques examine the total cell population in the sample and yield limited data on individual cells, which precludes study of cell cycle status of different cell types that may be present within the sample under analysis.
EP 798386 describes a method for the analysis of the cell cycle of cell sub-populations present in heterogeneous cell samples. This method uses sequential incubation of the sample with fluorescently labelled monoclonal antibodies to identify specific cell types and a fluorochrome that specifically binds to nucleic acids. This permits determination of the cell cycle distribution of sub-populations of cells present in the sample. However, as this method utilises flow cytometry, it still yields only non-dynamic data and requires serial measurements to be performed on separate samples of cells to determine variations in the cell cycle status of a cell population with time following exposure to an agent under investigation for effects on cell cycle progression.
A further disadvantage of flow cytometry techniques relates to the indirect, and inferred assignment of cell cycle position of cells based on DNA content. Since the DNA content of cell nuclei varies through the cell cycle in a reasonably predictable fashion, ie. cells in G2 or M have twice the DNA content of cells in G1, and cells undergoing DNA synthesis in S phase have an intermediate amount of DNA, it is possible to monitor the relative distribution of cells between different phases of the cell cycle. However, the technique does not allow precision in determining the cell cycle position of any individual cell due to ambiguity in assigning cells to G2 or M phases and to further imprecision arising from inherent variation in DNA content from cell to cell within a population which can preclude precise discrimination between cells which are close to the boundary between adjacent phases of the cell cycle. Additionally, variations in DNA content and DNA staining between different cell types from different tissues or organisms require that the technique is optimised for each cell type, and can complicate direct comparisons of data between cell types or between experiments (Herman, Cancer (1992), 69(6), 1553–1556). Flow cytometry is therefore suitable for examining the overall cell cycle distribution of cells within a population, but cannot be used to monitor the precise cell cycle status of an individual cell over time.
Cell cycle progression is tightly regulated by defined temporal and spatial expression, localisation and destruction of a number of cell cycle regulators which exhibit highly dynamic behaviour during the cell cycle (Pines, J., Nature Cell Biology, (1999), 1, E73–E79). For example, at specific cell cycle stages some proteins translocate from the nucleus to the cytoplasm, or vice versa, and some are rapidly degraded. For details of known cell cycle control components and interactions, see Kohn, Molecular Biology of the Cell (1999), 10, 2703–2734.
One of the most extensively characterised cell cycle regulators in human cells is cyclin B1, temporal and spatial expression and destruction of which controls cell transition from G2 to M and its exit from M. Cyclin B1 expression is driven by a cell cycle phase specific promoter which initiates expression at the end of S phase and peaks during G2. Once expressed, this protein constantly shuttles between the nucleus and the cytoplasm during the G2 phase, but it is primarily cytoplasmic because the rate of its export is much greater than its import. At the start of mitosis, cyclin B1 rapidly translocates into the nucleus, when its rate of import substantially increases, and its export decreases, in a phosphorylation dependent manner (FIG. 1). Thus, the localisation of cyclin B1 in the cell can be used to mark the transition from G2 phase to mitosis. Once a cell reaches metaphase, or, more accurately, when the spindle assembly checkpoint is satisfied, cyclin B1 is very rapidly degraded. Cyclin B1 destruction continues throughout the following G1 phase but stops once cells begin DNA replication. These events have been visualised in real time by micro-injection of fluorescently labelled cyclin B1 into living cells (Clute and Pines, Nature Cell Biology, (1999), 1, 82–87).
The controlling elements which regulate temporal expression and destruction have been elucidated in a number of studies. Biosynthesis of cyclin B1 has been shown to be controlled at the level of transcription by a promoter sequence that confines expression to the late S and G2 phases of the cell cycle (Piaggio et al, Exp. Cell. Research, (1995), 216, 396–402; Cogswell et al, Mol. Cell. Biology, (1995), 15, 2782–2790). Destruction of cyclin B1 at the appropriate time in M phase has been shown to be controlled by a 9 amino acid sequence, termed the destruction box (D-box) which targets the protein for proteolysis via ubiquitinylation. Expression of a Drosophila cyclin B-GFP fusion protein driven by a constitutive polyubiquitin promoter (Huang and Raff, EMBO Journal, (1999), 18(8), 2184–2195) has shown that fluorescently-tagged cyclin B mimics the behaviour of endogenous cyclin B in being degraded at the end of metaphase. Studies (Hagting et al, Current Biology, (1999), 9, 680–689) using human cyclin B1-GFP have shown that temporal changes in cytoplasmic and nuclear localisation of cyclin B1 with cell cycle progression is dependent on a nuclear export signal (NES), phosphorylation of which leads to nuclear import.
Other cell cycle checkpoints are similarly regulated by temporal and spatial control mechanisms and many of the components and interrelationships have been elucidated (Pines, J., Nature Cell Biology, (1999), 1, E73–E79).
A number of methods have been described which make use of certain components of the cell cycle control mechanisms to provide procedures which analyse or exploit cell proliferation status.
WO 00/29602 describes use of a cyclin A promoter to drive expression of GFP as a selectable marker for dividing transgenic stem cells to allow dividing cells to be isolated from a background of non-dividing cells by fluorescence activated cell sorting. While this method allows identification and selection of cells which have progressed past a certain stage in the cell cycle, it does not yield information on the cell cycle status of any given cell, other than historical information that the cell has or has not passed through the G2 phase of the cell cycle at some time in the past.
U.S. Pat. No. 6,048,693 describes a method for screening for compounds affecting cell cycle regulatory proteins, wherein expression of a reporter gene is linked to control elements which are acted on by cyclins or other cell cycle control proteins. In this method, temporal expression of a reporter gene product is driven in a cell cycle specific fashion and compounds acting on one or more cell cycle control components may increase or decrease expression levels. Since the assay system contains no elements which provide for the destruction of the reporter gene product nor for destruction of any signal arising from the reporter gene, the method cannot yield information on the cell cycle position of any cells in the assay and reports only on general perturbations of cell cycle control mechanisms.
U.S. Pat. No. 5,849,508 and U.S. Pat. No. 6,103,887 describe methods for determining the proliferative status of cells by use of antibodies which bind to cyclin A. These methods provide means for determining the percentage of proliferating cells in a test population relative to a control population.
U.S. Pat. No. 6,159,691 relates to a method for assaying for putative regulators of cell cycle progression. In this method, nuclear localisation signals (NLS) derived from the cell cycle phase specific transcription factors DP-3 and E2F-1 are used to assay the activity of compounds which act as agonists or antagonists to increase or decrease nuclear localisation of an NLS fused to a detectable marker.
A number of researchers have studied the cell cycle using traditional reporter molecules that require the cells to be fixed or lysed. For example Hauser and Bauer (Plant and Soil, 2000, 226, p 1–10) used β-glucuronidase (GUS) to study cell division in a plant meristem and Brandeis and Hunt (EMBO J., 1996, vol 15, pp 5280–5289) used chloramphenical acetyl transferase (CAT) fusion proteins to study variations in cyclin levels. Although these methods provide a means of studying the cell cycle position of a particular cell (using GUS) or the average cell cycle status of a population of cells (using CAT) both methods are destructive. Neither method allows the repeated analysis of a specific cell over time and they are therefore not suitable to follow the progression of a cell through the cell cycle.
None of the preceding methods, which use components of the cell cycle control mechanism, provide means for determining the cell cycle status of an individual cell or a population of cells. Consequently, methods are required that enable the cell cycle position of a single living cell to be determined non-destructively, allowing the same cell to be repeatedly interrogated over time, and which enable the study of the effects of agents having potentially desired or undesired effects on the cell cycle. Furthermore, it is desirable for cell cycle position to be determined from a probe controlled directly by cell cycle control components, rather than indirectly through DNA content or other indirect markers of cell cycle position as described above. The present invention describes a method which utilises key components of the cell cycle regulatory machinery in defined combinations to provide novel means of determining cell cycle status for individual living mammalian cells in a non-destructive process providing dynamic read out.
The present invention provides DNA constructs, and cell lines containing such constructs, that exhibit activation and destruction of a detectable reporter molecule in a cell cycle phase specific manner, by direct linkage of reporter signal switching to temporal and spatial expression and destruction of cell cycle components. This greatly improves the precision of determination of cell cycle phase status and allows continuous monitoring of cell cycle progression in individual cells. Furthermore, it has been found that key control elements can be isolated and abstracted from functional elements of the cell cycle control mechanism to permit design of cell cycle phase reporters which are dynamically regulated and operate in concert with, but independently of, endogenous cell cycle control components, and hence provide means for monitoring cell cycle position without influencing or interfering with the natural progression of the cell cycle.